ABSTRACT Vaccine adjuvants help stimulate the body to generate an immune response when co-injected with an antigen (the compound for which immunity is desired). Although the human body is capable of producing antibodies to most injected antigens, adjuvants can help boost the body?s ability to mount a successful and acquired immune response to the presented target. Adjuvants have been shown to enhance immunity against targets that usually exhibit poor immune responses?such as highly-purified and recombinantly-produced antigens. This is particularly important for individuals which may have a less robust immune system, such as children or the elderly. One of the well-studied adjuvants that is approved for use in humans nearly worldwide is the oil-in-water emulsion, MF59. The oil used in this formulation is the hydrocarbon triterpene, squalene. Squalene is a natural oil produced in all eukaryotic organisms as it is the precursor to sterols (e.g. cholesterol). MF59 is a component of Fluad, an FDA-approved seasonal influenza vaccine for people ?65 years old. The traditional source of squalene has been from shark liver oil, which is no longer viable nor sustainable. In light of these limitations, and the desire to develop more efficacious adjuvants, fermentable yeast and transgenic plants have been engineered to produce high amounts of squalene and several related, novel triterpenes. These include botryococcene plus mono- and di-methylated forms of squalene and botryococcene. The experiments proposed here are designed to accomplish two specific aims: (1) using a mouse model, demonstrate that our Synthetic Biology generated squalene performs equivalent to shark derived squalene in adjuvant vaccine formulations; and (2) evaluate our 5 novel triterpenes in adjuvant formulations for their abilities to induce elevated or altered immune responses compared to squalene. Our studies will quantitatively compare the immunogenicity of a trivalent influenza vaccine plus and minus the different triterpene adjuvant formulations by measuring antibody (IgG1, IgG2a and IgE) and hemagglutination inhibition (HI) titers. The possibility that our antigen-triterpene adjuvant formulations might induce undesirable effects will be assessed by measuring inflammatory cytokine levels in sera samples of the inoculated animals. Once the feasibility of using these compounds in an adjuvant system is demonstrated in this Phase I study, the foundation for several follow-on Phase II studies will be established: one, demonstrating the biological equivalency of our Synthetic Biology produced squalene would endorse efforts to use process engineering methodologies to scale-up our squalene manufacturing capabilities for marketplace commercialization; and two, if any of our novel triterpenes proves efficacious in stimulating antibody titers, we would need to pursue additional pre-clinical evaluations of their safety and potential for inciting undesirable inflammatory responses.